Gene targeting by homologous recombination is a powerful means to specifically modify a gene of interest. The availability of embryonic stem (ES) cells has been instrumental in the study of gene function in mice. In non-murine mammalian species, the lack of ES cells has been circumvented by gene targeting in primary somatic cells, followed by nuclear transfer. In spite of advances in molecular biology techniques, gene targeting in primary cells remains a challenge given the low frequency of homologous recombination (McCreath et al. (2000) Nature 405:1066-1069), the short lifespan of primary cells, and limitations in methods allowing the selection of properly targeted cells. Currently, primary cell gene targeting and production of offspring has mainly been performed with transcriptionally active genes, which are associated with a higher frequency of homologous recombination relative to silent genes (Denning et al. (2001) Nat. Biotechnol. 19:559-562). Furthermore, the selection of correctly targeted cells may be accomplished by having the targeted gene promoter drive the expression of a selection marker, a process which is not applicable to silent genes (Denning et al., supra; Lai et al. (2002) Science 295:1089-1092; Yifan et al. (2002) Nat. Biotechnol. 20:251-255; Thomson, A. J., et al., (2003) Reprod. Suppl. 61:495-508).
To fully evaluate the consequences of a genetic modification, both alleles of a gene need to be disrupted. In mice, this is generally accomplished by back crossing from a hemizygous transgenic founder animal to produce a homozygous targeted inbred line. Breeding to homozygosity is extremely time consuming in the mouse and represents an even more severe impediment in species that have a long generation interval and that are negatively impacted by the consequences of inbreeding. In the pig, two innovative approaches have been used to circumvent this limitation and produce homozygous β(1,3)-galactocyltransferase knockout animals. Hemizygous targeted primary cells were selected in vitro for the lack of the enzymatic activity resulting either from a spontaneous point mutation in the second allele of the gene (Denning et al., (2003) Reproduction 126:1-11) or for mitotic recombinants (Piedrahita (2000) Theriogenology 53: 105-16) and cloned offspring were made from the homozygous knockout cell lines. Unfortunately, these approaches are not generally useful for silent genes nor widely applicable for active genes. Thus, improved methods to study gene function in non-human mammals are desirable.
Antibody Production in Genetically Modified Animals
In 1890, Shibasaburo Kitazato and Emil Behring reported an experiment with extraordinary results; particularly, they demonstrated that immunity can be transferred from one animal to another by taking serum from an immune animal and injecting it into a non-immune one. This landmark experiment laid the foundation for the introduction of passive immunization into clinical practice. Today, the preparation and use of human immunoglobulin for passive immunization is standard medical practice. In the United States alone, there is a $1.4B per annum market for human immunoglobulin, and each year more than 16 metric tons of human antibody is used for intravenous antibody therapy. Comparable levels of consumption exist in the economies of most highly industrialized countries, and the demand can be expected to grow rapidly in developing countries. Currently, human antibody for passive immunization is obtained from the pooled serum of human donors. This means that there is an inherent limitation in the amount of human antibody available for therapeutic and prophylactic usage. Already, the demand exceeds the supply and severe shortfalls in availability have been routine.
In an effort to overcome some of the problems associated with the inadequate supply of human immunoglobulin, various technologies have been developed. For example, the production of human immunoglobulin by recombinant methods in tissue culture is routine. Particularly, the recombinant expression of human immunoglobulin in CHO expression systems is well known, and is currently utilized for the production of several human immunoglobulins and chimeric antibodies now in therapeutic use.
Mice retaining an unrearranged human immunoglobulin gene have also been developed for the production of human antibodies (e.g., monoclonal antibodies) (see, for example, PCT Publication Nos. WO98/24893; WO96/33735; WO 97/13852; WO98/24884; WO97/07671; and U.S. Pat. Nos. 5,877,397; 5,874,299; 5,814,318; 5,789,650; 5,770,429; 5,661,016; 5,633,425; 5,625,126; 5,569,825; and 5,545,806).
PCT Publication No. WO00/10383 describes modifying a human chromosome fragment and transferring the fragment into certain cells via microcell fusion. U.S. Pat. Nos. 5,849,992 and 5,827,690 describe the production of monoclonal antibodies in the milk of transgenic animals including mice, sheep, pigs, cows, and goats wherein the transgenic animals expressed human immunoglobulin genes under the control of promoters that provide for the expression of the antibodies in mammary epithelial cells. Essentially, this results in the expression of the antibodies in the milk of such animals, for example a cow. U.S. Patent Publication Nos. 2003-0037347-0 describe the expression of xenogenous human immunoglobulins in cloned, transgenic ungulates.
Notwithstanding the foregoing, further improved methods for producing ungulates that are amenable to being used as hosts for xenogenous antibody production are of great value to this industry.
Production of Prion Protein-deficient Bovines
The cellular prion protein PrPC is a ubiquitously expressed, plasma membrane, glycosylphosphatidylinositol-anchored glycoprotein. This protein plays a crucial role in the pathogenesis of transmittable spongiform encephalopathies such as BSE, in cattle, and Creutzfeldt-Jakob disease (CJD), in humans. Its disease-associated, protease-resistant isoform, PrPSc, has the ability to convert normal PrPC to PrPSc and is considered essential for the pathogenesis and transmittance of spongiform encephalopathies. Although the accumulation of PrPSc in neurons is associated with fatal neurodegeneration, the normal physiological function of the protein remains unclear. Mice with disruptions restricted to the coding region of the PrP gene have been generated and show only minor phenotypic deficits. Importantly, they are resistant to infection by PrPSc, suggesting that PrPC is necessary for pathogenesis of prion diseases (Prusiner et al. (1993) Proc. Natl. Acad. Sci. USA 90:10608-10612; Weissmann et al. (1994) Ann. NY Acad. Sci. 724:235-240).
BSE was first recognized in 1986 in the United Kingdom and now has been spread to many countries throughout the world. BSE can be transmitted from cattle to humans by direct consumption of contaminated beef products, resulting in a variant form of CJD (vCJD). Currently, there is no cure for the fatal disease. To reduce risk of exposure to the disease-causing PrPSc protein, expensive testing programs have been implemented and efforts to remove bovine components from a wide variety of products have been initiated in many countries. Cattle with homozygous null mutation in the PrP gene alleles could be used to alleviate concerns about BSE-contaminated bovine products. Furthermore, these animals could be useful as a model, in addition to the mouse, for investigating the involvement of PrP in BSE.